JP7802303B2 - Methods for Producing Cas3 Proteins - Google Patents

Description

The present invention relates to a method for producing Cas3 protein, and more specifically, to a method for producing recombinant Cas3 protein with high purity and high yield while maintaining activity.

Genome editing technology specifically cuts genomic DNA sequences within animal or plant cells and uses their inherent repair mechanisms to freely rewrite them into any sequence. Its use is expanding worldwide, not only in bioscience research but also in crop and livestock breeding, regenerative medicine, and gene therapy.

CRISPR-Cas systems found in bacteria and archaea are divided into Class 1, which cleaves target sequences using a complex of multiple proteins, and Class 2, which cleaves using a single protein. Genome editing tools developed to date, such as CRISPR-Cas9, CRISPR-Cas12 (Cpf1), and CRISPR-Cas13, are all classified as Class 2. However, it has recently been discovered that CRISPR-Cas3, a Type I CRISPR belonging to Class 1, can be used as a genome editing tool in eukaryotic cells (Patent Document 1). In particular, Type I-E CRISPR-Cas3 derived from the Escherichia coli K strain recognizes and binds to a 27-base target in addition to a 3-base PAM sequence, and has been shown to be able to efficiently introduce extensive deletion mutations of several hundred to several kb upstream of the target sequence in human cultured cells. Furthermore, compared to CRISPR-Cas9, the target recognition sequence in the guide RNA is longer, making non-specific cleavage less likely to occur, and therefore it is considered to be safer.

CRISPR-Cas3 is generally used by introducing it into cells as an expression plasmid and expressing it, but depending on the cell type, introduction and expression can be difficult, and sufficient genome editing efficiency may not be achieved. For this reason, it is desirable to introduce CRISPR-Cas3 into cells in the form of guide RNA (crRNA) and proteins (Cas3 protein and Cascade protein).

However, among the proteins that make up CRISPR-Cas3, it has been particularly difficult to prepare Cas3 protein in a practically usable form with sufficient quality and quantity. For example, existing Cas3 protein purification methods have been reported for the thermophilic bacterium Thermobifida fusca-derived Cas3 protein (TfuCas3) and the Escherichia coli K strain-derived Cas3 protein (EcoCss3) (Non-Patent Documents 1-3). However, because TfuCas3 is derived from a thermophilic bacterium, it is not suitable for use at the growth temperatures of many cells. On the other hand, EcoCas3 is derived from Escherichia coli and is active in a temperature range close to the growth temperatures of many cells, including animal cells, making it suitable for genome editing in a wide range of organisms with potential for industrial application. However, even when attempting to produce it using conventional methods, it has been difficult to purify it to high purity and high concentration while maintaining its activity (see Comparative Example 1 below).

International Publication No. 2018/225858

Huo Y. et al. , Nat. Struct. Mol. Biol. 2014 Sep;21(9):771-777 Mulepati S. & Bailey S. , J. Biol. Chem. 2013 Aug 2;288(31):22184-22192 Robert P. Hayes et al. , Nature 2016 530:499-503

The present invention was made in consideration of the problems inherent in the above-mentioned conventional technologies, and its purpose is to provide a method for producing an active form of Cas3 protein that can be used for genome editing in various organisms with high purity and high yield.

As a result of intensive research conducted by the inventors to solve the above-mentioned problems, they discovered that the Cas3 protein derived from E. coli has low thermal stability, and even when the recombinant protein is expressed by culturing it at the normal E. coli culture temperature, it is denatured and its activity is reduced.

Based on this unexpected finding, the inventors selected insect cells, which can be cultured at relatively low temperatures, introduced the Cas3 gene, and cultured them under various temperature conditions. They found that culturing at 20-28°C resulted in efficient expression of the Cas3 protein and maintained its activity.

Furthermore, the inventors investigated the purification conditions for recombinant Cas3 protein expressed in insect cells and found that by purifying in phosphate buffer, it is possible to recover active recombinant Cas3 protein in high purity and high yield.

Furthermore, the inventors discovered that the recombinant Cas3 protein recovered in this manner exhibits high activity even at the culture temperatures of animal cells and the like during the relatively short period required for genome editing processing, leading to the completion of the present invention.

In other words, the present invention relates to a method for producing recombinant Cas3 protein that can be used for genome editing in various cells at high purity and high concentration while maintaining its activity, and more specifically, provides the following inventions.

(1) A method for producing a Cas3 protein,
(a) culturing insect cells into which a Cas3 gene has been introduced at 20 to 28°C to express the Cas3 protein in the insect cells; and (b) recovering the expressed Cas3 protein.
A method comprising:

(2) The method described in (1), wherein the Cas3 protein is derived from Escherichia coli.

(3) The method described in (1) or (2), wherein the insect cells are Sf9 cells.

(4) A method described in any of (1) to (3), wherein the recovery of the expressed Cas3 protein includes purification of the Cas3 protein.

(5) The method described in (4), in which a tag is attached to the Cas3 protein and purification of the Cas3 protein includes affinity purification for the tag.

(6) The method described in (5), wherein the tag includes an HN tag.

(7) A method described in any of (4) to (6), wherein the purification of the Cas3 protein includes purification by gel filtration chromatography.

(8) A method described in any of (4) to (7), wherein the buffer used for purification is a phosphate buffer.

The present invention makes it possible to produce active recombinant Cas3 proteins with high purity and high yield. The method of the present invention can be used to produce various recombinant Cas3 proteins, but is particularly useful when applied to Cas3 proteins with low thermostability.

Furthermore, according to the present invention, it is possible to prepare the CRISPR-Cas3 system in a usable state before genome editing. Furthermore, it is possible to perform efficient genome editing even in cells in which it is difficult to introduce and express Cas3 as a gene, or in cells in which sufficient genome editing efficiency cannot be achieved when Cas3 is expressed as a recombinant protein. This makes it possible to use the CRISPR-Cas3 system simply and universally.

1 is a graph showing the purification of recombinant EcoCas3 protein expressed in E. coli by gel filtration chromatography. Electrophoresis photographs showing the expression of recombinant EcoCas3 protein in insect cells cultured at various temperatures. The graph (top) and SDS-PAGE photograph (bottom) show the results of purification of recombinant EcoCas3 protein expressed in insect cells by gel filtration chromatography using phosphate buffer. The lanes in the SDS-PAGE are as follows: a. supernatant, b. flow-through, c. TEV digestion, d. wash, e. backtrap flow-through, 2-20 SEC fractions, and f. concentrated fractions 13-16. 1 shows a graph (top) and an SDS-PAGE photograph (bottom) showing the results of purifying recombinant EcoCas3 protein expressed in insect cells by gel filtration chromatography using Hepes buffer. 1 is a graph showing the inflection point temperature due to thermal denaturation of recombinant EcoCas3 protein. The inflection point temperature was measured using TychoNT6. This is a graph showing the stability of EcoCas3, Cas9, Cas12, and TfuCas3 at 37° C. The stability of these proteins was measured by the change in fluorescence intensity that occurs when Sypro_orange binds to the hydrophobic region exposed to the solvent surface as the protein unfolds. FIG. 1 shows the plasmid for constructing the Multi-NLS Eco cascade. This is a capillary electrophoresis photograph showing the results of measuring genome editing activity in vitro using a complex of purified Cas3 protein, Cascade protein, and crRNA. The red arrow indicates detected DNA degradation. The left side shows the results using the EMX1-targeted cascade, and the right side shows the results using the Tyr-targeted cascade. [0023] Figure 1 shows the results of measuring genome editing activity in cultured human cells using a complex of purified Cas3 protein, Cascade protein, and crRNA. The top graph shows a representative image obtained by FACS analysis, and the bottom graph shows the analysis results of GFP knockout efficiency calculated from the number of GFP-negative cells (n = 3).

The present invention provides a method for producing a Cas3 protein.

In the present invention, the "Cas3 protein" is a protein that constitutes the CRISPR-Cas3 system and has nuclease activity and helicase activity. The Cas3 protein can cleave target DNA by cooperating with the cascade and crRNA that constitute the CRISPR-Cas3 system.

The type I-E CRISPR-Cas3 system, which is the most common type I CRISPR-Cas3 system, cuts DNA by crRNA working in cooperation with Cas3 and a cascade (Cse1 (Cas8), Cse2 (Cas11), Cas5, Cas6, and Cas7).

In Type I-A systems, the cascades are Cas8a1, Csa5 (Cas11), Cas5, Cas6, and Cas7; in Type I-B, the cascades are Cas8b1, Cas5, Cas6, and Cas7; in Type I-C, the cascades are Cas8c, Cas5, and Cas7; and in Type I-D, the cascade is Cas10d. The types I-F contain Csy1 (Cas8f), Csy2 (Cas5), Cas6, and Csy3 (Cas7) as cascade components, and the types I-G systems contain Cst1 (Cas8a1), Cas5, Cas6, and Cst2 (Cas7) as cascade components.

The Cas3 protein of the present invention may be of any origin, but a Cas3 protein derived from Escherichia coli is preferred because it is suitable for genome editing in a wide range of cells, including animal cells. The amino acid sequence of a typical Cas3 protein derived from Escherichia coli is shown in SEQ ID NO: 2, and the nucleotide sequence of the DNA encoding the protein is shown in SEQ ID NO: 1. The Cas3 protein of the present invention includes naturally occurring or artificially modified mutants.

The Cas3 protein of the present invention may be a protein consisting of an amino acid sequence that is highly identical to the amino acid sequence of the Cas3 protein derived from Escherichia coli set forth in SEQ ID NO: 1. High identity refers to, for example, sequence identity of 80% or more, preferably 85% or more, more preferably 90% or more (e.g., 91% or more, 92% or more, 93% or more, 94% or more), and even more preferably 95% or more (e.g., 96% or more, 97% or more, 98% or more, 99% or more). Sequence identity can be determined using tools such as BLAST (Basic Local Alignment Search Tool at the National Center for Biological Information) (e.g., using default, i.e., initial setting, parameters). Furthermore, the Cas3 protein of the present invention may be a protein consisting of an amino acid sequence in which one or more amino acids have been substituted, deleted, added, and/or inserted in the amino acid sequence of the Cas3 protein derived from Escherichia coli set forth in SEQ ID NO: 1. Here, "multiple" generally means 50 amino acids or less, preferably 30 amino acids or less, more preferably 20 amino acids or less, and particularly preferably 10 amino acids or less (for example, 5 amino acids or less, 3 amino acids or less, 2 amino acids or less, or 1 amino acid).

If necessary, functional molecules may be added to the Cas3 protein. Examples of functional molecules include, but are not limited to, nuclear localization signals that promote translocation into the nucleus of eukaryotic cells, tags that facilitate purification, and reporter proteins that facilitate detection. These functional molecules can be added, for example, to the N-terminus and/or C-terminus of the Cas3 protein.

Examples of nuclear localization signals include PKKKRKV (SEQ ID NO: 3) and KRTADGSEFESPKKRKV (SEQ ID NO: 4). Examples of tags include HN tags, His tags, FLAG tags, and glutathione-S-transferase (GST) tags. Examples of reporter proteins include fluorescent proteins such as green fluorescent protein (GFP) and chemiluminescent proteins such as luciferase.

In the production method of the present invention, insect cells into which the Cas3 gene has been introduced are cultured at 20 to 28°C, and the Cas3 protein is expressed in the insect cells (step (a)).

The well-known baculovirus expression system can be used, for example, to express recombinant Cas3 protein in insect cells. In one example of a method using the baculovirus expression system, the Cas3 gene is first cloned into a Bac-to-Bac vector such as pFastBac1, which is then introduced into E. coli containing baculovirus DNA to prepare baculovirus DNA incorporating the Cas3 gene. In addition to this method of preparing recombinant baculovirus DNA in E. coli, it is also possible to prepare it in insect cells. When using insect cells, a vector containing the Cas3 gene and baculovirus DNA are introduced into the insect cells, allowing homologous recombination to occur between the two. The prepared recombinant baculovirus DNA is then transfected into insect cells to prepare a recombinant baculovirus containing the Cas3 gene. The prepared recombinant baculovirus is then serially infected into insect cells to obtain a high-titer baculovirus, which is then used to infect insect cells to express the recombinant Cas3 protein. Suitable insect cells include, but are not limited to, Sf9 cells.

Insect cells are preferably cultured at 20-28°C to express recombinant Cas3 protein. Below 20°C, the expression efficiency of recombinant Cas3 protein tends to decrease, while above 28°C, the expressed recombinant Cas3 protein tends to denature during the culture process. From the perspective of further suppressing denaturation, 20-24°C is more preferable, 20-22°C is even more preferable, and 20°C is particularly preferable. There are no particular restrictions on the culture time as long as it is sufficient for the expression of the recombinant Cas3 protein, but it is usually 24 hours or more, preferably 60-72 hours.

In the manufacturing method of the present invention, the expressed Cas3 protein is then recovered (step (b)).

Various protein separation and purification methods can be used to recover the expressed Cas3 protein. Cell disruption and centrifugation can be used to isolate recombinant Cas3 protein from cells. For example, cells can be disrupted by sonication, then centrifuged at 100,000 g, and the supernatant can be collected to obtain a soluble fraction containing recombinant Cas3 protein.

When a tag is attached to the Cas3 protein, affinity purification of the tag can be used to purify the recombinant Cas3 protein. For example, if the tag is an HN tag or His tag, affinity purification can be performed using a nickel column; if the tag is a FLAG tag, affinity purification can be performed using beads bound to an antibody against the FLAG tag; and if the tag is a glutathione-S-transferase (GST) tag, affinity purification can be performed using glutathione Sepharose. From the perspective of electrically neutralizing the Cas3 protein and suppressing aggregation, an HN tag is preferred as the tag to be attached to the Cas3 protein.

In addition, the purification of the recombinant Cas3 protein in the present invention preferably includes purification by gel filtration chromatography. Because the Cas3 protein is a globular protein with a molecular weight of approximately 100 kDa, it is preferable to select a column with a fractionation range appropriate for globular proteins of that molecular weight. Commercially available columns such as Superdex 200 Increase (Cytiva) can be used as such columns.

The buffer used for purification is preferably phosphate buffer, as it prevents aggregation due to denaturation of the Cas3 protein.

The recombinant Cas3 protein prepared in this manner possesses excellent activity and can exert its activity without denaturation, even at temperatures of 37°C, for the relatively short period of time required for genome editing, within the several hours required for genome editing. Indeed, in this example, excellent DNA cleavage activity and high genome editing efficiency were observed at 37°C. Therefore, the recombinant Cas3 protein obtained by the method of the present invention, when combined with a Cascade protein and crRNA, can efficiently perform genome editing in a wide range of cells.

The present invention will be explained in more detail below based on examples and comparative examples, but the present invention is not limited to the following examples.

Comparative Example 1: Preparation of EcoCas3 protein using Escherichia coli Conventional methods for producing EcoCas3 (Mulepati S. & Bailey S., J. Biol. Chem. 2013 Aug. 2; 288(31): 22184-22192) have the following drawbacks: (i) Escherichia coli containing an EcoCas3-encoding plasmid must be cultured at a low temperature of 20°C; (ii) EcoCas3 must contain maltose-binding protein (MBP) or small ubiquitin-like enzymes to maintain its solubility. There were many problems with this method, such as (i) the need to fuse a SUMO modifier to the EcoCas3 protein, (ii) the need to co-express the HtpG protein, a chaperone molecule, (iii) the yield was a maximum of 1 mg per 1 L of culture, and (v) the difficulty in producing highly pure EcoCas3 because bands of proteins other than EcoCas3 were observed in the electrophoresis pattern. In fact, when EcoCas3 protein was prepared using E. coli according to the above literature, it was found to be of low purity and low yield (Figure 1), and its activity was also extremely low.

[Example 1] Preparation of EcoCas3 protein using insect cells Sf9 When a protein derived from a prokaryote is expressed as a recombinant protein in a higher eukaryote, post-translational modifications that do not occur in prokaryotes may occur. For this reason, production of EcoCas3 protein in eukaryotes has not been attempted. However, in view of the problems described in Comparative Example 1, the present inventors attempted to prepare EcoCas3 protein using insect cells Sf9.

(1) Construction of a plasmid for expression of recombinant EcoCas3 protein. An 8HN tag (a tag formed by fusing a His tag and an HN tag via a GS linker / SEQ ID NO: 5) and NLS (SEQ ID NO: 3) were fused to the N-terminus of EcoCas3, and an NLS (SEQ ID NO: 3) was also fused to the C-terminus to synthesize a gene (SEQ ID NOs: 6, 7). The HN tag (a repeat sequence of histidine and asparagine) was used to neutralize the positive charge of the His tag, which has the potential to aggregate the target protein. To confirm expression, a gene was also created in which EGFP was fused to the 3' end of the 8HN tag as a reporter (SEQ ID NOs: 8, 9).

The above fusion gene was cloned into the pFastbac-1 plasmid (ThermoFishers). The resulting EcoCas3/pFastbac-1 plasmid was transformed into DH10bac and integrated into the baculovirus genome within DH10bac by homologous recombination. The baculovirus genome containing the EcoCas3 gene was then extracted.

(2) Expression of Recombinant EcoCas3 Protein Baculovirus genomes containing the EcoCas3 gene or the EGFP-fused EcoCas3 gene were transfected into Sf9 cells, and baculoviruses containing the EcoCas3 gene were produced in the Sf9 cells. Sf9 cells were then passaged with these baculoviruses to obtain high-titer viruses for EcoCas3 expression. Sf9 cells were infected with these high-titer viruses, and EcoCas3 was expressed as a recombinant protein.

To investigate the expression of recombinant EcoCas3 protein, a baculovirus containing the EGFP-fused EcoCas3 gene was used. Sf9 cells were infected with the baculovirus at 28°C for 24 hours, and then the Sf9 cells were cultured at various temperatures (12°C to 28°C) for 60 hours to express the recombinant EcoCas3 protein. The cells were sonicated and centrifuged at 100,000g. The collected supernatant (soluble fraction) was subjected to electrophoresis and fluorescence detection.

As a result, we confirmed the expression of recombinant EcoCas3 protein in the soluble fraction at temperatures above 16°C, with the expression efficiency in the soluble fraction being particularly high at temperatures above 20°C (Figure 2). In the following experiments, we used recombinant EcoCas3 protein expressed in Sf9 cells at 20°C.

(3) Preparation of Recombinant EcoCas3 Protein Recombinant EcoCas3 protein carrying a His-tag-like 8HN tag was affinity purified using a nickel column, and then final purification was performed using gel filtration chromatography.

Insect cells expressing the recombinant EcoCas3 protein were sonicated, centrifuged at 100,000 g, and the collected supernatant (soluble fraction) was mixed with nickel agarose resin (Qiagen) to bind the recombinant EcoCas3 protein to the resin _. The recombinant EcoCas3 protein was then eluted from the resin using a wash buffer (20 mM HEPES or 20 mM _KH2PO4 , 350 mM NaCl, 40 mM imidazole, 0.5 mM DTT, pH 7.0) and an elution buffer (20 mM Hepes or 20 mM _KH2PO4 , 350 mM NaCl, 200 mM imidazole, 0.5 mM DTT, pH _7.0 ).

The eluted fraction was digested with TEV to cleave the 8HN tag attached to the N-terminus of the recombinant EcoCas3 protein. Finally, the recombinant EcoCas3 protein was purified by gel filtration chromatography. A Superdex 200 _increase (Cytiva) column was used for purification, and the mobile phase buffer used was "20 mM HEPES or 20 mM _KH2PO4 , 200 mM NaCl, 1.0 mM DTT, pH 7.0."

As a result, when purification was performed using HEPES buffer, a large amount of void fraction containing EcoCas3, which is thought to have aggregated during gel filtration chromatography, was confirmed (Figure 3). On the other hand, when purification was performed using phosphate buffer, the inflection point temperature (Ti) improved by approximately 2°C, and ultimately, up to 2 mg of purified EcoCas3 protein was successfully obtained per 1 L of culture (Figure 4). It is believed that the use of phosphate buffer suppressed the amount of EcoCas3 separated into the void fraction. When purity was confirmed by SDS-PAGE, no protein bands other than the target EcoCas3 protein were detected, demonstrating that the purified EcoCas3 protein was significantly more pure than conventional EcoCas3 purified protein using an E. coli expression system.

[Example 2] Measurement of the thermal stability of recombinant EcoCas3 protein The thermal stability of recombinant EcoCas3 protein was evaluated by measuring the inflection point temperature (Ti) of the thermal denaturation profile using a TychoNT6 (NanoTempar). The peak shift of autofluorescence derived from intramolecular tryptophan residues accompanying thermal unfolding of the protein molecule was detected at two wavelengths, 330 nm and 350 nm, and the inflection point temperature (Ti) of the thermal denaturation profile was determined by plotting the ratio of the fluorescence intensities against temperature.

We also measured the thermal stability of the recombinant EcoCas3 protein using the SYPRO Orange fluorescent reagent. SYPRO Orange emits fluorescence by binding to the denatured hydrophobic regions of the protein. The increase in SYPRO Orange binding and fluorescence intensity, which depend on the exposure of the hydrophobic regions of the protein to solvent due to structural changes associated with thermal denaturation, were detected using a real-time PCR device. Fluorescence detection was performed at an excitation wavelength of 473 nm and an emission wavelength of 520 nm.

These results demonstrate that while the inflection point temperature of the recombinant EcoCas3 protein was similar to that of Cas9 in measurements using TychoNT6 (Figure 5), stability measurements at a constant temperature of 37°C using SYPRO Orange fluorescent reagent revealed that the recombinant EcoCas3 protein completely denatured after approximately 8 hours (Figure 6). These results suggest that the recombinant EcoCas3 protein is in a state of high entropy, which indicates molecular disorder, and exhibits large fluctuations. Therefore, the Gibbs free energy change, a thermodynamic parameter, is small, making it more unstable than Cas9, Cas12, and TfuCas3. These results regarding the stability of the recombinant EcoCas3 protein clearly explain the highly efficient expression of the protein in Sf9 cells at temperatures lower than the typical insect cell culture temperature (28°C) (see the results in Example 1(2)). On the other hand, it was found that the purified recombinant Cas3 protein retains high activity for several hours even at 37°C, which clearly explains why it can be used without any particular problems in cleaving target DNA in vitro or in various cells (results of Examples 3 and 4 described below).

Example 3: Preparation of Eco Cascade Complex In order to simultaneously introduce the recombinant Eco Cascade into cells and the recombinant EcoCas3 protein to perform genome editing, it is important to efficiently transport it into the nucleus within the cell. However, the molecular weight of the Eco Cascade is large, approximately 0.4 MDa, raising concerns about its rate of nuclear transport. Therefore, the inventors attempted to introduce more NLS into the cascade by splitting the five-gene operon that constitutes the Eco Cascade into two and adding NLS to the 3' end of each operon, thereby achieving highly efficient transport into the cell nucleus.

The Eco cascade is a supramolecular complex consisting of Cas8-Cas11-Cas7-Cas5-Cas6. It consists of one Cas8 molecule, two Cas11 molecules, five Cas7 molecules, two Cas5 molecules, and one Cas6 molecule. The inventors constructed three plasmids: a His-tag-fused Cas11-NLS in the pCDFuet-1 plasmid; a "Cas8-Cas11-Cas7 operon" with an NLS added to the 3' end; and a "Cas5-Cas6 operon" with an NLS added to the 3' end in the pRSFDuet-1 plasmid; and crRNA in the pACYCDuet-1 plasmid (Figure 7, SEQ ID NOs: 10-24).

These three plasmids were then introduced into E. coli JM109(DE3) (a lysogenic E. coli strain harboring bacteriophage λDE3 incorporating a T7 RNA polymerase gene under the control of the lacUV5 promoter), and recombinant proteins or crRNA were expressed using IPTG. The Multi-NLS-Eco cascade was affinity purified using a nickel column via the His tag introduced into Cas11, followed by gel filtration chromatography. As a result, approximately 1 mg of Multi-NLS-Eco cascade was successfully produced from a 2 L culture.

The target sequences for crRNA were sequences within the human EMX1 gene, the mouse Tyr gene, and the Aequorea victoria GFP gene.

Example 4: In vitro DNA cleavage assay. The target DNA cleavage activity of purified EcoCas3 and EcoCascade proteins was investigated in vitro using double-stranded DNA. Cas3 protein (20 nM), crRNA-Cascade complex (20 nM), and double-stranded DNA (60 ng/μL) containing the target sequence were mixed in a reaction buffer (5 mM HEPES-K pH 7.5, 60 mM KCl, ₁₀ mM MgCl , ₁₀ μM CoCl , 2.5 mM ATP). The reaction solution was incubated at 37°C for 1 hour and subjected to capillary electrophoresis using a MultiNa (Shimadzu Corporation). The target sequences were the EMX1 gene region and the Tyr gene region. For EMX1, we also prepared and examined a sequence in which the PAM sequence of the double-stranded DNA was changed from "AAG," which can be recognized by the Escherichia coli-derived Type IE CRISPR, to "CCA," which cannot be recognized.

As a result, when double-stranded donor DNA containing the AAG target sequence, which can be recognized by the PAM sequence, was mixed, degradation of the donor DNA was observed (Figure 8). On the other hand, when donor DNA modified to CCA, which cannot recognize the PAM sequence, was mixed, no DNA degradation was observed. When the Cas3 protein alone or the Cascade complex alone was used, no DNA cleavage activity was observed. On the other hand, when the Cascade complex alone was used, a band was detected above normal. This is thought to be because the Cascade complex recognizes and binds to the DNA, causing it to become larger than normal under non-denaturing conditions, resulting in a gel shift.

Example 5: Activity measurement in human cultured cells HEK293T. Using reporter HEK293T cells carrying mCherry-P2A-EGFP, the mutagenesis efficiency of purified EcoCas3 and EcoCascade proteins in human cells was examined. Cas3 protein (30 μM or 45 μM), a complex of GFP-targeted crRNA and the Cascade (30 μM or 45 μM) was introduced into reporter cells by electroporation using a Neon Transfection System (Thermo Fisher Scientific). After culturing the cells for 5 days at ₃₇ °C and 5% CO2, all cells were harvested and the number of GFP-negative cells was counted using an SH800 (Sony) to calculate the mutagenesis efficiency.

As a result, approximately 20% of cells were GFP-negative when transfected at 30 μM, and approximately 40% when transfected at 45 μM (Figure 9). This demonstrates that genome editing using the CRISPR-Cas3 protein can be performed with high efficiency, with a maximum of approximately 40%.

The Cas3 protein produced by the method of the present invention can be used for genome editing of various cells, and therefore can be used not only in basic research but also in various applied fields of genome editing technology, such as medicine, agriculture, and industry.

Claims (7)

A method for producing a Cas3 protein, comprising:
(a) culturing insect cells into which a Cas3 gene has been introduced at 20 to 24 °C to express the Cas3 protein in the insect cells; and (b) recovering the expressed Cas3 protein.
Including,
The Cas3 protein is derived from Escherichia coli;
method. The method of claim 1 , wherein the insect cells are Sf9 cells. The method of claim 1 or 2 , wherein the recovery of the expressed Cas3 protein comprises purifying the Cas3 protein. The method of claim 3 , wherein a tag is attached to the Cas3 protein and purification of the Cas3 protein comprises affinity purification against the tag. The method of claim 4 , wherein the tag comprises an HN tag. The method of any one of claims 3 to 5 , wherein the purification of the Cas3 protein comprises purification by gel filtration chromatography. The method according to any one of claims 3 to 6 , wherein the buffer used for purification is a phosphate buffer.